Whole proteome tiling microarrays

ABSTRACT

The present invention relates to a microarray comprising at least 50,000 oligopeptide features per cm 2  where the oligopeptide features represent at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the proteome of a virus or an organism. The present invention further relates to methods for the synthesis of such microarrays and methods of using microarrays comprising at least 50,000 oligopeptide features per cm 2 . In an embodiment of the invention, the oligopeptide features represent proteins expressed in the same species, wherein the oligopeptide features are presented in a tiling pattern representing at least about 5,000 to-at least about 25,000 proteins expressed in a species. In some embodiments, the oligopeptide microarray features represent proteins expressed in the same species, wherein the microarray features are present in a tiling pattern that represents at least about 5,000 to at least about 50,000 expressed proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication 61/454,214, filed Mar. 18, 2011, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a microarray comprising at least 50,000oligopeptide features per cm² where the oligopeptide features representat least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or 100% of the proteome of a virus or anorganism. The present invention further relates to methods for thesynthesis of such microarrays and methods of using microarrayscomprising at least 50,000 oligopeptide features per cm². In anembodiment of the invention, the oligopeptide features representproteins expressed in the same species, wherein the oligopeptidefeatures are presented in a tiling pattern representing at least about5,000, at least about 10,000, at least about 15,000, at least about20,000, or at least about 25,000 of the proteins expressed in a species.In some embodiments, the oligopeptide microarray features representproteins expressed in the same species, wherein the microarray featuresare present in a tiling pattern that represents between about 5,000 and50,000 of the proteins expressed in a species, between about 10,000 and50,000 of the proteins expressed in a species, between about 15,000 and50,000 of the proteins expressed in a species, between about 20,000 and50,000 of the proteins expressed in a species, or between about 25,000and 50,000 of the proteins expressed in a species.

BACKGROUND OF THE INVENTION

Oligopeptide microarrays are widely used in research and healthcare.Within these areas, oligopeptide microarrays are suitable for manydifferent applications. Oligopeptide microarrays for example provide atool for the identification of biologically active motifs, e.g.oligopeptide microarrays may imitate potential active motifs of ligandsfor screening the binding to corresponding receptors. Furthermore, theoligopeptide microarrays might reflect specific sequences of diseaseassociated antigens. Such oligopeptide microarrays can be utilized todetect antibodies from patient samples suggesting the presence ofcertain inflammatory diseases, infections, and the like. Anotherimportant application of the oligopeptide microarrays is the discoveryof biochemical interactions, including the binding of proteins or DNA.Oligopeptide microarrays can further be used for the profiling ofcellular activity, the activity of enzymes, the adhesion of cells, andthe like.

Traditional methods for the analysis of autoimmune diseases involves thedetection of autoantibodies and include enzyme linked immunosorbentassays (ELISAs), Western blot analysis, immunoprecipitation analysis andflow-based assays. Routine assays for detection of autoantibodies isgenerally performed by ELISAs and fluorescence assays. Individual assaysare performed in microtiter plates, with a single antigen per well.These tests are performed one-at-a-time, are laborious, and expensive.Oligopeptide arrays have been used to characterize and detectautoantibodies, but they have generally utilized purified antigenmolecules spotted onto substrates. The antigens must be produced inrecombinant expression systems and purified, which is a time-consumingprocess. These antigens are generally whole proteins, or known antigenicdomains, and do not allow the characterization of specific epitopes.Synthetic peptide arrays have been utilized as well, however theproduction of these peptides is done by commercial automated peptidesynthesizers, and then spotted onto slides. However, they cannot achievethe scale of peptides synthesized by maskless array synthesis (MAS)technology.

Traditional methods, such as ELISA, are laborious and costly, and canonly be done one antigen at a time. While spotted oligopeptidemicroarrays are available, and allow parallel detection of multipleautoantibodies, the cost of producing those arrays is very expensive dueto the cost of producing purified antigen molecules in a recombinantexpression system. In addition they have a very low resolution andcannot achieve the comprehensive coverage of substantially the wholeproteome that an oligopeptide microarray can. Many proteins cannot besynthesized in in vitro systems, which would prevent their use on sucharrays.

Furthermore, antigens that are expressed and then spotted onto amicroarray often only represent a small percentage of the full proteinsequence. Antibodies in one patient may target one set of antigenicdomains, which in another patient, the antibodies may target acompletely different set of antigenic domains in the same protein. Suchpatient-to-patient differences could arise from misfolding of proteins,a common problem in autoimmune disease, thus causing differentialpresentation of protein domains to antibody producing B cells.Oligopeptide arrays are thus preferred because they allow all possibleantigenic sites within a given protein to be examined in order to detectpatterns or fingerprints across many patients.

The object of the present invention is the provision of microarrays witha high density oligopeptides with improved capabilities for highresolution analysis (including, but not limited to, serologicalanalysis), a method for their synthesis and their use. The advantage ofthe microarrays according to the invention is their oligopeptide densityand the coverage of substantially the whole proteome of an organism bythe application of a tiling concept. Because of this oligopeptidedensity, the microarrays according to the invention allow the paralleldetection of all autoantibodies in a human serum sample with a singlebinding assay. In addition, specific information about the location ofepitopes is obtained from the assay by the introduction of the tilingconcept. Therefore, the present invention provides a simple,cost-effective method for screening for a wide variety of autoimmunediseases, as well as rapid custom epitope mapping, screening peptidesfor small molecule binding, synthesis of antibody-like arrays forprotein expression analysis, proteome-scale peptide scanning, and manymore applications.

SUMMARY OF THE INVENTION

The present invention relates to a microarray with high density ofoligopeptide features, thereby allowing for the detection of proteininteractions across an organism's proteome. An embodiment of theinvention is a microarray comprising at least 50,000 oligopeptidefeatures per cm². Another embodiment is a microarray having oligopeptidefeatures representing at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 99%, or 100% of the proteomeof a target selected from a virus or organism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a synthetic process useful in the present invention,wherein a digital micromirror device is utilized for maskless arraysynthesis of oligopeptide arrays.

FIG. 2 depicts a representative “tiling” of poly(A) polymerase alpha(PAPOLA) sequence from amino acid 620 to amino acid 649 (SEQ ID NO:1).

FIG. 3 provides examples of polyclonal anti-PAPOLA binding to “targeted”peptide array (FIG. 3A), the same array viewing binding to the fulllength PAPOLA protein (FIG. 3B), and binding to PAPOLA target on a fullproteome array (FIG. 3C).

FIG. 4 shows a summary of autoantibodies and epitopes determined throughbinding experiments.

FIG. 5 demonstrates a sensitivity titration of anti-PAPOLA antibodydilution.

FIG. 6 shows binding of an isolated IgG pool from a colorectal cancerserum sample to an array with approximately 40,000 12-mer peptides withan 11-mer overlap.

FIG. 7 provides examples of polyclonal anti-ADA binding to “targeted”peptide array (FIG. 7A), the same array viewing binding to the fulllength ADA protein (FIG. 7B), and binding to ADA target on a fullproteome array (FIG. 7C).

FIG. 8 demonstrates binding of monoclonal anti-poly-Histidine on a fullproteome array.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are set forth to illustrate and define themeaning and scope of various terms used to describe the inventionherein.

The term “microarray” as used herein refers to a two dimensionalarrangement of features on the surface of a solid or semi-solid support.Features as used herein are defined areas on the microarray comprisingbiomolecules, such as peptides, nucleic acids, carbohydrates, and thelike. The features can be designed in any shape, but preferably thefeatures are designed as squares or rectangles. The features can exhibitany density of biomolecules. In some cases, the density is at least10,000 features per cm².

The term “proteome” as used herein refers to all proteins or a set ofproteins expressed by a genome, cell, tissue, or organism, including allproteins as contained in currently existing databases describing theexpressed proteins of a particular organism, and further includes allvariants or a set of variants of proteins resulting from alternativesplicing of genes, all post-translationally modified proteins, andproteins translated from genes containing one or more single nucleotidepolymorphisms (SNPs), frame shift mutations, deletions, inversions, andthe like. Examples of existing databases describing the expressedproteins of various organisms are:

UniProt (Universal Protein Resource; uniprot.org on the World Wide Web);

Ensembl (ensembl.org on the World Wide Web);

VEGA (Vertebrate Genome Annotation; vega.sanger.ac.uk/ on the World WideWeb);

CCDS (Consensus CDS; ncbi.nlm.nih.gov/projects/CCDS/ on the World WideWeb);

UCSC Genome Browser (genome.ucsc.edu on the World Wide Web);

Protein database at NCBI (ncbi.nlm.nih.gov/protein on the World WideWeb); and

RCSB Protein Data Bank (pdb.org/ on the World Wide Web).

Such databases can be queried regarding particular organisms. Forexample, the UniProt database can be queried for proteins by taxonomy.For determination of proteins containing SNPs, one of skill in the artwould look to currently existing databases containing informationregarding SNPs, such as NCBI's dbSNP (ncbi.nlm.nih.gov/projects/SNP/ onthe World Wide Web).

The term “posttranslational modification” as used herein refers to achemical modification of a protein, the modification occurs after thetranslation of the protein. Posttranslational modifications include butare not limited to glycosylation, phosphorylation, acetylation,methylation, palmitoylation, amidation, and the like.

The term “serological response” as used herein refers to the productionof antibodies within an organism, for example, within the human body,wherein the antibodies are directed against certain antigens. Theantibodies can be directed against foreign antigens, such as moleculesor structures on the surface of intruded molecules, compounds ormicroorganism. Preferably, the antibodies can be directed against theorganism's own antigens (in instances of autoimmune diseases,precancerous lesions or cancer). Preferably, the organism's own antigensare proteins. The serological response can be measured by diagnostictests, detecting the antibodies specific for the response in bodyfluids, preferably in serum, thereby giving information about the reasonof the response in order to institute therapeutic actions. As diagnostictests, test can be used known by the skilled person, such as ELISA,Western Blot, Agglutination, and the like.

The term “external stimulus” as used herein refers to stimuli inducing aserological response, whereas the stimuli have their origin outside theorganism, preferably, the human body. External stimuli include but arenot limited to microorganisms, pollen, peptides, proteins, poisons, andthe like.

The term “autoimmune reaction” as used herein refers to malfunctions ofthe immune system, preferably the human immune system. Such malfunctionsare characterized by the production of autoantibodies directed againstthe organism's own antigens or by the production of immune cells thattarget and attack particular cells or tissues of the body, preferablythe human body. The autoimmune reaction can result in symptomsconstituting an autoimmune disorder. The definition of autoimmunereaction includes, but is not limited to, immune reactions that occur inreaction to the presence of preneoplastic lesions, neoplastic lesions,malignant cells, malignant tissues.

The term “solid support” as used herein refers to any solid material,having a surface area to which organic molecules can be attached throughbond formation or absorbed through electronic or static interactionssuch as covalent bond or complex formation through a specific functionalgroup. The support can be a combination of materials such as plastic onglass, carbon on glass, and the like. The functional surface can besimple organic molecules but can also comprise of co-polymers,dendrimers, molecular brushes and the like.

The term “plastic” as used herein refers to synthetic materials, such ashomo- or hetero-co-polymers of organic building blocks (monomer) with afunctionalized surface such that organic molecules can be attachedthrough covalent bond formation or absorbed through electronic or staticinteractions such as through bond formation through a functional group.Preferably the term “plastic” refers to polyolefin, which is a polymerderived by polymerization of an olefin (e.g., ethylene propylene dienemonomer polymer, polyisobutylene). Most preferably, the plastic is apolyolefin with defined optical properties, like TOPAS® or ZEONOR/EX®.

The term “light transmission” as used herein refers to the property ofmatter, whereby the matter is transparent to a certain extent such thatlight can pass through the matter. The amount of light passing throughis dependent on the extent of transparency or transmittance.

The term “spatially resolved photoirradiation” as used herein refers tothe fact that light is directed precisely onto defined areas of asurface, preferably the surface of a microarray, by a device, such as anarray of individually addressable aluminum micro mirrors. The devicecontrols the overall pattern of light projected on the surface, therebypreparing the areas for the next coupling reaction. Preferably, lightexposure leads to the cleavage of photolabile protecting groups and theun-masking of functional groups within the areas where the nextcomponent, e.g., an amino acid or a nucleotide, is to be coupled. Thissystem is in parallel combined with a synthesizer in order to producemicroarrays. By using this technique, that is directing light toindividually addressable aluminum micro mirrors, 385,000 to 4.2 millionunique probe features can be synthesized on a single microarray ofmicroscope-slide size of 75×25 mm.

The term “maskless photolithography” as used herein refers to atechnique for the synthesis of DNA or oligopeptide microarrays withoutthe use of photo-masks. In maskless photolithography a device is usedfor directing light onto a defined area of a surface, preferably thesurface of a microarray, in order to induce photo reactions, preferablythe release of photolabile protecting groups. Examples for such a devicecan be a micro mirror device, a light-transmissive LCD display or a beamsplitter. Preferably, the device is an array of individually addressablealuminum mirror elements that are operable under software control. Suchmirror elements individually direct light onto a defined area of asurface, preferably the surface of a microarray. A preferred micromirror device is the Digital Light Processor (DLP) from TexasInstruments, Inc.

The term “protecting group” as used herein refers to a substituent,functional group, ligand, or the like, which is cleavable bound (e.g.,via covalent bond, ionic bond, or complex) to a potentially reactivefunctional group and prevents the potentially reactive functional groupfrom reacting in an uncontrolled manner. Preferably, the protectinggroup is cleavable bound via a covalent bond. The protecting group canbe cleaved off the respective reactive functional group by any fashion,such as by acids, bases, fluoride, enzymes, reduction or oxidation.Preferably, the protecting group is cleaved off by light exposure.Protecting groups according to the invention are photo labile protectinggroups, which include, but are not limited to, o-nitrobenzyl-oxycarbonyl(NBOC), o-nitrophenyl-ethoxycarbonyl (NPEOC),2-(3,4-methylenedioxy-2-nitrophenyl)-propyloxy-carbonyl (MeNPPOC),2-(3,4-methylenedioxy-2-nitrophenyl)-oxycarbonyl (MeNPOC),2-(2-nitrophenyl)-propoxycarbonyl (NPPOC),2-(2-nitro-4-benzoylphenyl)-2′-propyl-1′-oxycarbonyl (Benzoyl-NPPOC),dimethoxy-benzo-inylyl-oxycarbonyl (DMBOC),2-(2-nitrophenyl)-ethylsulfonyl (NPES), (2-nitrophenyl)-propylsulfonyl(NPPS), and the like.

The term “functional group” as used herein refers to any of numerouscombinations of atoms that form parts of chemical molecules, thatundergo characteristic reactions themselves, and that influence thereactivity of the remainder of the molecule. Typical functional groupsare hydroxyl, carboxyl, aldehyde, carbonyl, amino, azide, alkynyl, thioland nitril. Potentially reactive functional groups include, for example,amines, carboxylic acids, alcohols, double bonds, and the like.Preferred functional groups are potentially reactive functional groupsof amino acids such as amino groups or carboxyl groups.

The term “natural amino acid” as used herein refers to one of the 20amino acids used for protein biosynthesis as well as other amino acidswhich can be incorporated into proteins during translation (includingpyrrolysine and selenocysteine). The 20 natural amino acids includehistidine, alanine, valine, glycine, leucine, isoleucine, aspartic acid,glutamic acid, serine, glutamine, asparagine, threonine, arginine,proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine andlysine.

The term “non-natural amino acid” as used herein refers to an organiccompound that is not among those encoded by the standard genetic code,or incorporated into proteins during translation. Therefore, non-naturalamino acids include amino acids or analogs of amino acids, but are notlimited to, the D-isostereomers of amino acids, the beta-amino-analogsof amino acids, citrulline, homocitrulline, homoarginine,hydroxyproline, homoproline, ornithine, 4-amino-phenylalanine,cyclohexylalanine, α-aminoisobutyric acid, N-methyl-alanine,N-methyl-glycine, norleucine, N-methyl-glutamic acid, tert-butylglycine,α-aminobutyric acid, tert-butylalanine, 2-aminoisobutyric acid,α-aminoisobutyric acid, 2-aminoindane-2-carboxylic acid,selenomethionine, dehydroalanine, lanthionine, γ-amino butyric acid, andderivatives thereof wherein the amine nitrogen has been mono- ordi-alkylated.

The term “peptide” or “oligopeptide” as used herein refers to organiccompounds composed of amino acids, which are arranged in a linear chainand joined together by peptide bonds between the carboxyl and aminogroups of adjacent amino acid residues. The term “peptide” or“oligopeptide” preferably refers to organic compounds composed of lessthan 70 amino acid residues, more preferably of less than 35 amino acidresidues, more preferably of less than 25 amino acid residues.

The term “amino group” as used herein refers to primary (—NH₂), orsecondary (—NHR₁) amino groups. Examples of amino groups include, butare not limited to, —NH₂, —NHCH₃, —NHC(CH₃)₂. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

The term “reactive amino group” as used herein refers to an amine thatcan react with a functional group to form a covalent bond between thenitrogen of the amino group and the electrophile of the functionalgroup, such as a peptide bond.

The term “polar organic solvent” as used herein refers to solvents whichare water soluble in that a homogeneous mixture of the solvent in wateris possible at room temperature under ambient conditions. Preferredpolar organic solvents are methanol, ethanol, propanol, methyl ethylketone, acetonitrile, acetone, tetrahydrofuran (THF), dioxane,dimethylsulfoxide (DMSO), n-methyl-2-pyrrolidone (NMP),dimethylformamide (DMF), dimethylacetamide (DMA).

The term “base” as used herein refers to a substance capable ofaccepting a proton in polar or non-polar solvents. The base of choicefor a particular reaction depends on the starting materials, the solventand the temperature used for a specific reaction. Examples of basesinclude carbonate salts, phosphates, halides, hydroxides, hydrides,heterocyclic amines, disilylamides, trialkylamines, bicyclic amines,alkali metal hydrides, nitrogen-containing bases.

The term “synthesis cycle” as used herein refers to a predeterminednumber of successive reaction steps which are conducted to perform asynthesis of oligopeptides. Preferably the term “synthesis cycle” refersto a predetermined number of successive reaction steps which areconducted during solid phase synthesis of oligopeptides in order toattach the respective next amino acid to the previous functional group.Oligopeptide synthesis comprises a predetermined number of synthesiscycles, wherein in each cycle one specific amino acid is attached to theprevious functional group. Therefore, the number of the cycles dependson the number of amino acids of the oligopeptide. For example, for thesynthesis of a peptide micro array containing 20 amino acid buildingblocks, 20 cycles are required to elongate each feature of the peptidemicroarray by one amino acid residue. The combination of amino acidresidues within an oligopeptide depends on the specific amino acidswhich are attached one after another to the respective previousfunctional group during the successive synthesis cycles.

The term “scavenger molecule” as used herein refers to an agent reactivewith free radicals. Also, a scavenger molecule can be a molecule thatreacts with olefins by means of an addition reaction as known in thefield of peptide chemistry. Scavenger molecule according to theinvention is an agent, which can be contained in polar organic solventsin order to react with side products of the deprotection step. Scavengermolecules include but are not limited to strong nucleophilic amines likepiperidine, piperazine, imidazole and the like as well as radicalquenchers, such as hydroxylamine, TEMPO, Oxo-TEMPO, sterically hinderedphenols, and thiophenols.

Oligopeptide arrays of the present invention provide a number of usesnot currently available with respect to existing oligopeptide arraytechnology. As an example, such oligopeptide microarrays are useful inantibody detection related to autoimmune diseases. In general,autoimmune diseases result from an overactive immune response of thebody to its own tissues and/or substances. The body attacks its owncells, resulting in various disease symptoms. More than 80 differentautoimmune diseases are known. The symptoms of the different autoimmunediseases vary depending on the disease itself as well as theconstitution of the patient's immune system. Some symptoms, however,might be identical between different autoimmune diseases. Symptomsinclude but are not limited to: fatigue, malaise, dizziness, high bodytemperature or fever, increased sensitivity to temperature in hands andfeet. Severe symptoms are inflammation, weakness and stiffness ofmuscles and joints, digestive or gastrointestinal problems resulting inweight changes, blood sugar changes, abnormal blood pressure,irritability, anxiety or depression, reduced libido, infertility andchange in size of an organ or even the destruction of an organ ortissue. The diversity of the symptoms and the difficult classificationimpedes effective diagnosis and therapeutic approaches. The success of atherapy of autoimmune diseases generally depend on an individual'ssymptoms, results from physical examination and diagnostic tests, thelatter being an essential element of effective therapeutic approaches.Therefore simple, cost-effective methods of screening for a wide varietyof autoimmune diseases are required.

Furthermore, oligopeptide microarrays are suitable for the analysis ofbasically any other disease involving interactions of proteins, whichcan be represented on an array. For example, oligopeptide microarraysprovide a tool for the identification of biologically active motifsinvolved in the onset of certain diseases, e.g., oligopeptidemicroarrays may imitate potential active motifs of ligands for screeningthe binding to corresponding receptors. Furthermore, the oligopeptidemicroarrays might reflect specific sequences of disease associatedantigens. Such oligopeptide microarrays can be utilized to detectantibodies from patient samples suggesting the presence of certaininflammatory diseases, infections, and the like. Another importantapplication of the oligopeptide microarrays is the discovery ofbiochemical interactions, including DNA-protein-orprotein-protein-interactions. Oligopeptide microarrays can further beused for the profiling of cellular activity, the activity of enzymes,the adhesion of cells, and the like.

Autoimmune applications are just one example of the utility ofhigh-density oligopeptide arrays. Other applications include, but arenot limited to, rapid custom epitope mapping, screening peptides forsmall molecule binding, synthesis of antibody-like arrays for proteinexpression analysis, proteome-scale peptide scanning, and many moreapplications.

Different methods for the production of oligopeptide microarrays can beused in the present invention. Spotting prefabricated peptides orin-situ synthesis by spotting reagents, e.g., on membranes, are twopotential approaches. One of the most commonly used methods to generatepeptide arrays of higher density are the so-called photolithographictechniques, where the synthetic design of the desired biopolymers iscontrolled by suitable photolabile protecting groups (PLPG) releasingthe linkage site for the respective next component (amino acid,oligonucleotide) upon exposure to electromagnetic radiation, preferablylight (Fodor et al., Nature 364 (1993) 555-556, Fodor et al., Science251(1991) 767-773).

Two different photolithographic techniques are: 1) A photolithographicmask is used to direct light to specific areas of the synthesis surfaceeffecting localized deprotection of the PLPG. The drawback of thistechnique is that a large number of masking steps are required resultingin a relatively low overall yield and high costs, e.g., the synthesis ofa peptide of only six amino acids in length could require over 100masks. 2) The second technique is the so-called masklessphotolithography, where light is directed to specific areas of thesynthesis surface effecting localized deprotection of the PLPG bydigital projection technologies, such as micro mirror devices(Singh-Gasson et al., Nature Biotechn. 17 (1999) 974-978). Thus, timeconsuming and expensive production of exposure masks is unnecessary.

Synthesis cycle steps useful for the manufacture of oligopeptidemicroarrays of the present invention are exemplified in FIG. 1. In oneembodiment, plastic solid supports comprising reactive amino groupsand/or reactive ε-amino-hexanoic-acid linker moieties are irradiatedusing light from a light source directed to a digital micromirror device(DMD) which redirects the light onto the surface of the solid support.The illumination of a particular location using the DMD is either “on”or “off,” depending upon whether a particular feature site shouldincorporate the next amino acid in the synthesis cycle. If theoligopeptide desired at a particular location requires such nextsuccessive amino acid, light reflected from the DMD at that particularlocation will be “on” (i.e., that particular feature will beilluminated), thus cleaving the protecting group from the amino acidpresent at that site. If the oligopeptide desired at a particularlocation should not incorporate the next successive amino acid, the DMDwill be “off” in that position (i.e., that feature will not beilluminated), and the amino acid present at that site will remainprotected.

In FIG. 1, four particular microarray features (features 1, 2, 3, and 4,respectively) are exemplified. Each feature is occupied by a protectedamino acid from a previous synthesis cycle step. Features 2 and 4 areilluminated by the DMD, cleaving the protecting group from therespective amino acids at those features. The next successive aminoacid, asparagine with a photolabile protecting group in this example, isthen allowed to flow over the microarray and will incorporate into theactivated features 2 and 4, but will not incorporate into the blockedfeatures 1 and 3. Thus, the oligopeptides being constructed on features2 and 4 are elongated by the addition of blocked asparagine, while theoligopeptides being constructed at features 1 and 3 remain unchanged. Inthe next synthesis cycle step as exemplified in FIG. 1, features 1 and 4are illuminated by the DMD, cleaving the protecting group from therespective amino acids at those features, while features 2 and 3 remainprotected. Protected alanine is then allowed to flow over themicroarray, and incorporates into features 1 and 4 but does notincorporate into features 2 and 3. Successive steps continue until theappropriate oligopeptides are synthesized at all four features.

In certain methods, amino acids introduced in the synthesis ofoligopeptide microarrays are protected with NPPOC or MeNPOC,respectively. The method using NPPOC protected amino acids has thedisadvantage that the half-life of all (except one) protected aminoacids is within the range of approximately 2 to 3 minutes under certainconditions. In contrast, under the same conditions, NPPOC-protectedtyrosine exhibits a half-life of almost 10 minutes. As the velocity ofthe whole synthesis process depends on the slowest sub-process, thisphenomenon increases the time of the synthesis process by a factor of 3to 4. Concomitantly, the degree of damage by photogenerated radical ionsto the growing oligomers increases with increasing and excessive lightdose requirement. Amino acids, which are protected with Benzoyl-NPPOC,show a half-life of approximately 2 to 3 minutes, includingBenzoyl-NPPOC-protected tyrosine. The drawback of using exclusivelyBenzoyl-NPPOC-protected amino acids is that the dissociated protectinggroups adhere to the solid support, thereby inhibiting the synthesisprocess of the oligopeptides, a phenomenon that is unknown for DNAarrays and is being attributed to light induced addition of the releasedbenzophenone moiety to either the growing peptide chain or the solidsupport itself. In protein array technology, plastic derived supportsare preferred due to their low non-specific binding to proteins samples.However, benzophenone moieties are well known in the state of the art toadd to hydrocarbons as present in plastic surfaces by a radicalmechanism.

In one aspect of the invention, the oligopeptides of each feature canhave a length of between 9 and 18 amino acid residues. In some cases,the oligopeptide features are between 10 and 15 amino acids in length.In another embodiment, the length of the oligopeptides is 12 amino acidresidues. In one embodiment, substantially all of the oligopeptidefeatures are the same length. For example, at least 90%, at least 95%,at least 99%, or 100% of the oligopeptide features can be the samelength in a microarray provided herein. In some cases, the sequences ofthe oligopeptide features of a microarray provided herein aresubstantially identical. In a further aspect of the invention, the aminoacid sequence of each feature overlaps the amino acid sequence of atleast one other feature by at least 3, at least 6, or at least 9contiguous amino acid residues. In one embodiment, the sequence of eachfeature has an overlap of n−1 with at least one other feature, whereinn=the length of the oligopeptide of the at least one other feature.

In one aspect of the invention, each oligopeptide feature represents aportion of a target proteome, where the target is selected from a virusand an organism. Organisms appropriate for the microarrays and methodsprovided herein can include mammalian species (e.g., human, mouse),plant species (e.g., Arabidopsis), insect species (e.g., Drosophila),prokaryotes, yeast, and fungi.

In an embodiment of the invention, the oligopeptide features representproteins expressed in the same species, wherein the oligopeptidefeatures are presented in a tiling pattern representing at least 5,000of the proteins expressed in a species, at least 10,000 expressedproteins, at least 15,000 expressed proteins, at least 20,000 expressedproteins, or at least 25,000 expressed proteins. In some embodiments,the oligopeptide features represent proteins expressed in the samespecies, wherein the oligopeptide features are present in a tilingpattern that represents between about 5,000 and 50,000 expressedproteins, between about 10,000 and 50,000 expressed proteins, betweenabout 15,000 and 50,000 expressed proteins, between about 20,000 and50,000 expressed proteins, or between about 25,000 and 50,000 expressedproteins.

In another aspect of the invention, the oligopeptide features representproteins expressed in a mammalian proteome, wherein the oligopeptidefeatures are present in a tiling pattern that represents at least 5,000mammalian proteins, at least 10,000 mammalian proteins, at least 15,000mammalian proteins, or at least 20,000 mammalian proteins. In someembodiments, the oligopeptide features represent proteins expressed in amammal, wherein the oligopeptide features are present in a tilingpattern that represents between about 5,000 and 50,000 mammalianproteins, between about 10,000 and 50,000 mammalian proteins, betweenabout 15,000 and 50,000 mammalian proteins, between about 20,000 and50,000 mammalian proteins, or between about 25,000 and 50,000 mammalianproteins.

In another aspect of the invention, the oligopeptide features representproteins expressed in the human proteome, wherein the oligopeptidefeatures are present in a tiling pattern that represents at least 5,000human proteins, at least 10,000 human proteins, at least 15,000 humanproteins, or at least 20,000 human proteins. In some embodiments, theoligopeptide features represent proteins expressed in human, wherein theoligopeptide features are present in a tiling pattern that representsbetween about 5,000 and 50,000 human proteins, between about 10,000 and50,000 human proteins, between about 15,000 and 50,000 human proteins,between about 20,000 and 50,000 human proteins, or between about 25,000and 50,000 human proteins.

In another aspect of the invention, the oligopeptide features representproteins expressed in the mouse proteome, wherein the oligopeptidefeatures are present in a tiling pattern that represents at least 5,000mouse proteins, at least 10,000 mouse proteins, at least 15,000 mouseproteins, or at least 20,000 mouse proteins. In some embodiments, theoligopeptide features represent proteins expressed in mouse, wherein theoligopeptide features are present in a tiling pattern that representsbetween about 5,000 and 50,000 mouse proteins, between about 10,000 and50,000 mouse proteins, between about 15,000 and 50,000 mouse proteins,between about 20,000 and 50,000 mouse proteins, or between about 25,000and 50,000 mouse proteins.

In another embodiment, the oligopeptide features represent proteinsexpressed in a plant proteome, wherein the oligopeptide features arepresent in a tiling pattern that represents at least 5000 plantproteins, at least 10,000 plant proteins, at least 15,000 plantproteins, at least 20,000 plant proteins, or at least 25,000 plantproteins. In some embodiments, the oligopeptide microarray featuresrepresent proteins expressed in plant, wherein the oligopeptide featuresare present in a tiling pattern that represents between about 5,000 and50,000 plant proteins, between about 10,000 and 50,000 plant proteins,between about 15,000 and 50,000 plant proteins, between about 20,000 and50,000 plant proteins, or between about 25,000 and 50,000 plantproteins.

In another embodiment, the oligopeptide features represent proteinsexpressed in the Arabidopsis proteome, wherein the oligopeptide featuresare present in a tiling pattern that represents at least about 5,000Arabidopsis proteins, at least 10,000 Arabidopsis proteins, at least15,000 Arabidopsis proteins, at least 20,000 Arabidopsis proteins, or atleast 25,000 Arabidopsis proteins. In some embodiments, the oligopeptidefeatures represent proteins expressed in Arabidopsis, wherein theoligopeptide features are present in a tiling pattern that representsbetween about 5,000 and 50,000 Arabidopsis proteins, between about10,000 and 50,000 Arabidopsis proteins, between about 15,000 and 50,000Arabidopsis proteins, between about 20,000 and 50,000 Arabidopsisproteins, or between about 25,000 and 50,000 Arabidopsis proteins.

In another embodiment, the oligopeptide features represent proteinsexpressed in an insect proteome, wherein the oligopeptide features arepresent in a tiling pattern that represents at least 5,000 insectproteins, at least 10,000 insect proteins, or at least about 15,000insect proteins. In some embodiments, the oligopeptide featuresrepresent proteins expressed in insect, wherein the oligopeptidefeatures are present in a tiling pattern that represents between about5,000 and 30,000 insect proteins, between about 10,000 and 30,000 insectproteins, between about 15,000 and 30,000 insect proteins, between about20,000 and 30,000 insect proteins, or between about 25,000 and 30,000insect proteins.

In another embodiment, the oligopeptide features represent proteinsexpressed in the Drosophila proteome, wherein the oligopeptidemicroarray features are present in a tiling pattern that represents atleast 5000 Drosophila proteins, at least 10,000 Drosophila proteins, orat least about 15,000 Drosophila proteins. In some embodiments, theoligopeptide microarray features represent proteins expressed inDrosophila, wherein the oligopeptide features are present in a tilingpattern that represents 5,000 to 50,000 Drosophila proteins, 10,000 to50,000 Drosophila proteins, 15,000 to 50,000 Drosophila proteins, 20,000to 50,000 Drosophila proteins, or 25,000 to 50,000 Drosophila proteins.

In another aspect of the invention, the oligopeptide features representproteins expressed in prokaryotes, wherein the oligopeptide features arepresent in a tiling pattern that represents at least 2000 prokaryoteproteins, at least 3,000 prokaryote proteins, at least 4,000 prokaryoteproteins, or at least 5,000 prokaryote proteins. In some embodiments,the oligopeptide features represent proteins expressed in prokaryotes,wherein the oligopeptide features are present in a tiling pattern thatrepresents between about 2,000 and 10,000 prokaryote proteins, betweenabout 3,000 and 10,000 prokaryote proteins, between about 4,000 and10,000 prokaryote proteins, or between about 5,000 and 10,000 prokaryoteproteins.

In another aspect of the invention, the oligopeptide features representproteins expressed in E. coli, wherein the oligopeptide features arepresent in a tiling pattern that represents at least 2000 E. coliproteins, at least 3,000 E. coli proteins, at least 4,000 E. coliproteins, or at least 5,000 E. coli proteins. In some embodiments, theoligopeptide features represent proteins expressed in E. coli, whereinthe oligopeptide features are present in a tiling pattern thatrepresents between about 2,000 and 10,000 E. coli proteins, betweenabout 3,000 and 10,000 E. coli proteins, between about 4,000 and 10,000E. coli proteins, or between about 5,000 and 10,000 E. coli proteins.

In another aspect of the invention, the oligopeptide features representproteins expressed in fungi, wherein the oligopeptide features arepresent in a tiling pattern that represents at least 2000 fungiproteins, at least 3,000 fungi proteins, at least 4,000 fungi proteins,or at least 5,000 fungi proteins. In some embodiments, the oligopeptidefeatures represent proteins expressed in fungi, wherein the oligopeptidefeatures are present in a tiling pattern that represents between about2,000 to 10,000 fungi proteins, between about 3,000 to 10,000 fungiproteins, between about 4,000 to 10,000 fungi proteins, or between about5,000 to 10,000 fungi proteins.

In another aspect of the invention, the oligopeptide features representproteins expressed in yeast, wherein the oligopeptide features arepresent in a tiling pattern that represents at least 2000 yeastproteins, at least 3,000 yeast proteins, at least 4,000 yeast proteins,or at least 5,000 yeast proteins. In some embodiments, the oligopeptidefeatures represent proteins expressed in yeast, wherein the oligopeptidefeatures are present in a tiling pattern that represents between about2,000 to 10,000 yeast proteins, between about 3,000 to 10,000 yeastproteins, between about 4,000 to 10,000 yeast proteins, or between about5,000 to 10,000 yeast proteins.

The present invention also concerns a method for the synthesis of anoligopeptide microarray as described in the previous paragraphs. Inseveral embodiments, the overlap of each oligopeptide feature withanother feature is exactly 9 amino acids on microarrays producedaccording to the method of the invention.

The present invention further concerns the use of an oligopeptidemicroarray for serological analysis. In some cases, a microarraydescribed herein is used for analysis of a serological response to anexternal stimulus. In another embodiment, a microarray described hereinis used for detecting an autoimmune reaction.

One aspect of the present invention is an oligopeptide microarraycomprising at least 50,000 features per cm², characterized in that thefeatures represent at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 99%, or 100% of the proteomeof a target proteome, where the target is selected from a virus or anorganism. In one embodiment the microarray comprises oligopeptidefeatures representing the human proteome, where the oligopeptidefeatures represent at least 90%, at least 95%, at least 99%, or 100% ofthe human proteome.

Another aspect of the present invention are high density microarraysthat have extensive features in a compact area. Embodiments of thepresent microarrays can have a variety of oligopeptide featuredensities. For example, the microarrays of the present inventioncomprise at least 10,000 oligopeptide features/cm², 50,000 oligopeptidefeatures/cm², at least 100,000 oligopeptide features/cm², at least200,000 oligopeptide features/cm², at least 300,000 oligopeptidefeatures/cm², at least 400,000 oligopeptide features/cm², at least500,000 oligopeptide features/cm², or at least 1,000,000 oligopeptidefeatures/cm². Further, certain embodiments of microarrays have featuredensity within a variety of feature density ranges. For example, thedensity can comprise a range of 10,000 to 1,000,000 oligopeptidefeatures/cm², 50,000 to 1,000,000 oligopeptide features/cm², 100,000 to1,000,000 oligopeptide features/cm², 200,000 to 1,000,000 oligopeptidefeatures/cm², 300,000 to 1,000,000 oligopeptide features/cm², 400,000 to1,000,000 oligopeptide features/cm², 500,000 to 1,000,000 oligopeptidefeatures/cm², 10,000 to 500,000 oligopeptide features/cm², 50,0000 to500,000 oligopeptide features/cm², 100,000 to 500,000 oligopeptidefeatures/cm², 200,000 to 500,000 oligopeptide features/cm², or any rangefound within a lower level of 10,000 and an upper level of 1,000,000oligopeptide features/cm².

In one embodiment, the microarray has a density of at least 10,000oligopeptide features per cm² and, in some cases, at least 50,000oligopeptide features per cm². Applied to the area of a microarray, asingle microarray of 75×25 mm size may contain at least 385,000 uniqueoligopeptide features, at least 720,000 unique oligopeptide features, orat least 2.1 million unique oligopeptide features.

In another embodiment, oligopeptides of each feature can have basicallyany length on the array. In some cases, the oligopeptides have a lengthof between 2 and 50 amino acid residues. I some cases, the oligopeptidescan have a length of between 5 and 25 amino acid residues or between 9and 18 amino acid residues.

In another embodiment, substantially all of the oligopeptides are thesame length. In some cases, substantially all of the oligopeptides arebetween 2 and 50 amino acid residues in length, between 5 and 25 aminoacid residues in length, between 10 and 15 amino acid residues inlength, and in certain embodiments, are 12 amino acid residues long.

In yet another embodiment, the sequence of each feature has an overlapof at least 3, at least 6, and or at least 9 contiguous amino acidresidues with the amino acid sequence of at least one other feature. Inone embodiment, the sequence of each feature has an overlap of n−1 withat least one other feature, wherein n=the length of the oligopeptide ofthe at least one other feature.

In the present invention, protein sequences representing either theentire proteome or specific autoimmune disease targets are tiled on theoligopeptide microarray according to the invention and an antibodybinding assay performed using human sera. Fluorescently labeledsecondary antibodies can then be bound to the oligopeptide microarray todetect which oligopeptides on the microarray the antibodies in the serahave bound. One of skill in the art will appreciate that other methodsfor detection of binding are also available, such as (but not limitedto) direct labeling of the binding antibody. Direct labeling methodswould be particularly suited when the present invention is used toinvestigate the binding of monoclonal antibodies. Further, it isunderstood that the present invention is not necessarily limited toantibodies, but can be applied to antibody fragments, or other bindingpartners that specifically target a protein or peptide.

Oligopeptides can be tiled for protein sequences in various ways,depending on the array platform and number of protein targets. Asexamples, for a full proteome design utilizing the 2.1M platform, 12-meroligopeptides at a 3 amino acid step, or 16-mer peptides at a 6 aminoacid step, can be used. The former would allow the characterization ofall 9-mer epitopes in the proteome, and the latter would allow thecharacterization of all 10-mer epitopes.

For the present invention, the construction of the oligopeptidemicroarray involves the collection of the protein sequences representingthe proteome of a virus or an organism, for example, the human proteome.Respective information is often publicly available. Peptides areproduced from the protein sequences by grabbing substrings from theprotein sequence. The substrings are 12 to 16 amino acids in length, andare selected at 3 to 6 amino acid spacing, illustrating the tilingconcept of the present invention. A simple compression algorithm,identical to the one used for DNA synthesis is used to either discard,or truncate, peptide sequences that would take too long to produce. Thepeptide sequences are laid out in an array design using software, suchas the ArrayScribe software, and mask files are produced for the 3P MASunit using a custom perl script analogous to the method used to produceDNA array masks. Arrays are synthesized on a MAS unit, and processed ina manner identical to other peptide arrays. Binding can be done usingmixers and following protocols known by the skilled person. Detection ofthe signal via fluorescently labeled secondary antibodies is done viamethods known by the skilled person. Scanning and quantification ofimages can be accomplished using MS200 scanner and NimbleScan software.

FIG. 2 provides an example of the tiling concept utilized in certainembodiments of the present invention. The top line of FIGS. 2A-2C eachshow the PAPOLA protein sequence from amino acid 620 to amino acid 649(the “PAPOLA sequence”). In FIG. 2A, a set of 4 probe features isillustrated beneath the PAPOLA sequence that embodies a set of 12-meroligopeptides wherein the probes in each feature differs by one aminoacid (a 1×12 array). Thus, a set of probe features configured as a 1×12array will have an overlap of 11 amino acids between a particularfeature and at least one other feature. One alternative tilingembodiment is illustrated in FIG. 2B. Again, the top line shows thePAPOLA sequence; however, the tiling arrangement of the 4 illustratedprobe features is designed so that each feature has 12-mer probes thatdiffer by 3 amino acids (a 3×12 array). Thus, a set of probe featuresconfigured as a 3×12 array will have an overlap of 9 amino acids betweena particular feature and at least one other feature. In FIG. 2C, anotherembodiment is illustrated wherein the probe features are again 12-mers,but the “step” between features is 6 amino acids (a 6×12 array). In suchan array, a set of probe features configured as a 6×12 array will havean overlap of 6 amino acids between a particular feature and at leastone other feature.

Another aspect of the present invention is a method for the synthesis ofan oligopeptide microarray as described above. In one embodiment, amethod is used wherein the overlap of each feature in amino acidsequence with the amino acid sequence of another feature is between 2and 40 amino acid residues, between 5 and 20 amino acids, between 8 and15 amino acids, or, in some cases, exactly 9 amino acids.

In another aspect, the present invention provides methods for usingoligopeptide microarrays. For example, microarrays of the presentinvention are useful in antibody detection related to autoimmunediseases. A microarray as described above can be used for any analysisof suitable tissue or body fluid, preferably for serological analysis.One embodiment is the use of an oligopeptide microarray as describedabove for the analysis of a serological response to internal or externalstimuli, preferably external stimuli. Another embodiment is the use ofan oligopeptide microarray as described above for detecting anautoimmune reaction. In some cases, an oligopeptide microarray can beused for characterization of the antigen binding capacity of anantisera, an antibody, or a fragment of an antibody. For example, amicroarray can be used for characterization of the antigen bindingcapacity of a monoclonal antibody or a polyclonal antisera.

The NPPOC-protected amino acids were synthesized by adding therespective amino acid to a Na₂CO₃ solution in H₂O. To that solutiontetrahydrofurane (THF) was added subsequently. Afterwards, a solution of2-nitrophenyl-2-propan-1-ol (NPPOC—Cl) in THF was added. THF was removedin a rotary evaporator under vacuum and the residue was extracted. Theresidue was acidified and extracted with ethylacetate. Extracts werewashed with H₂O and evaporated in vacuo to dryness. The residue wasdissolved in dichloromethane and purified by column chromatography.

The microarray according to the invention can be synthesized accordingto the following steps:

-   a) providing a plastic solid support-   b) coupling to the plastic solid support an amino acid which is    protected at its amino group with NPPOC or a derivative thereof-   c) optionally capping unreacted amino acids-   d) optionally washing the plastic solid support-   e) deprotecting the amino acid by photoirradiation at 350 to 410 nm-   f) repeating steps b) to e) for a predetermined number of times.

Alternatively, the microarray according to the invention can besynthesized according to the following steps:

-   a) providing a plastic solid support, the solid support having a    primary or secondary amine coupled to the surface-   b) coupling to the plastic solid support an amino acid which is    protected at its amino group with NPPOC or a derivative thereof-   c) optionally, capping sites that did not couple the amino acid    derivative of the previous step-   d) optionally washing the plastic solid support-   e) site selectively deprotecting the amino acid by photoirradiation    at 350 to 410 nm, the selection being provided by a mask or a    mask-free device, preferably in a polar organic solvent, most    preferably containing a scavenger molecule to react with side    products of this deprotection step-   f) repeating steps b) to e) for a predetermined number of times.

Alternatively, the microarray according to the invention can besynthesized according to the following steps:

-   a) providing a plastic solid support, the solid support having a    primary or secondary amine coupled to the surface-   b) coupling to the plastic solid support an amino acid which is    protected at its amino group with NPPOC or a derivative thereof,    forming a peptide bond to the solid support-   c) optionally, capping sites that did not couple the amino acid    derivative of the previous step-   d) optionally, washing the solid support-   e) site selectively deprotecting the amino acid by photoirradiation    at 350 to 410 nm, that selection being provided by a mask or a    mask-free device, preferably in a polar organic solvent, most    preferably containing a scavenger molecule to react with side    products of this deprotection step-   f) repeating steps b) to e) for a predetermined number of times-   g) deprotecting all “permanent protection groups” located at the    side-chains of amino acids, e.g. Lysine(e-amino-BOC)—-   h) optionally, treating the peptide microarray with a reducing agent    in order to reverse oxidative damage occurring at Cysteine- or    Methionine-sulfur.

The support of the microarray can be made of any material known by theskilled person used for the synthesis of a microarray, preferably thesupport is made of plastic, glass, carbon on glass, metal on glass,plastic on glass. Preferably, plastic is used as a support. Mostpreferred is a plastic solid support. More preferably, the supportcomprises a surface layer and a body, wherein the body consists ofpolyolefin. More preferred is that the surface of the support comprisesreactive amino groups. More preferably, ε-amino-hexanoic-acid is coupledto the surface of the support. The support can be provided in any shape,such as beads, gels, plates, membranes, slides or preferably chips. TheC-terminal amino acid residues can be bound to the surface of thesupport, preferably a plastic solid support, via peptide bonds. TheC-terminal amino acids of the oligopeptides can be coupled to thesurface of the support, preferably a plastic solid support, withε-amino-hexanoic-acid.

The surface of the support can comprises functional groups, capable offorming bonds, such as peptide bonds. Preferably the surface of thesupport can be coated with a respective compound, which then providesthe functional groups, capable of forming the bonds. The support can becoated with ε-amino-hexanoic-acid or ε-amino-hexanoic-acid, which iscoupled to the surface of the support.

The first amino acid, which is coupled to the support and the followingamino acids coupled thereto are protected by any protecting groupcapable of preventing the potentially reactive functional group of theamino acid from reacting under certain reaction conditions. Preferredprotecting groups are o-nitro-benzyloxy-carbonyl (NBOC),o-nitrophenyl-ethoxycarbonyl (NPEOC),2-(3,4-methylenedioxy-2-nitrophenyl)-propyloxy-carbonyl (MeNPPOC),2-(3,4-methylenedioxy-2-nitrophenyl)-oxycarbonyl (MeNPOC),dimethoxy-benzo-inylyl-oxycarbonyl (DMBOC),2-(2-nitrophenyl)-ethylsulfonyl (NPES) and(2-nitrophenyl)-propylsulfonyl (NPPS). Most preferred protecting groupsare 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC), or derivatives thereof.Preferably the used protecting groups are NPPOCs and/or NPPOCderivatives. Preferably, the derivatives are2-(2-nitro-4-benzoyl-phenyl)-propoxycarbonyl (NPPOC), Benzoyl-NPPOCs.

Any natural or non-natural amino acid protected by the above mentionedprotecting groups can be used for the synthesis of peptide microarrays.Preferably, natural amino acids are used for the synthesis ofoligopeptide microarrays. The amino acids can be protected by NPPOCsand/or NPPOC derivatives, such as Benzoyl-NPPOC. 16-19 different aminoacids, such as histidine, alanine, valine, glycine, leucine, isoleucine,aspartic acid, glutamic acid, serine, glutamine, asparagine, threonine,arginine, proline, phenylalanine, tryptophan, cysteine, tyrosine,methionine and lysine, which are protected with NPPOCs and/or NPPOCderivatives are used. Some amino acids can be protected withBenzoyl-NPPOC, preferably tyrosine is protected with Benzoyl-NPPOC.

Protecting groups are cleavable bound to potentially reactive functionalgroups of amino acids in order to prevent the potentially reactivefunctional groups from reacting in an uncontrolled manner. Theprotecting groups are preferably cleavable bound to the amino acids by acovalent binding. The protecting groups can be cleaved off therespective functional group by any fashion, such as by acids, bases,fluoride, enzymes, reduction or oxidation. Preferred is the use ofphotolabile protecting groups, which are cleaved off by light exposureor irradiation, respectively.

Irradiation can be used for cleaving off the photolabile protectinggroups, which spans the whole spectrum of electromagnetic radiation.Preferred for cleaving off the photolabile protecting group is the rangefrom UV- to the IR-light, ranging approximately from 200 nm to 700 nm.More preferred deprotection is performed at 200 nm to 400 nm.Deprotection can be performed at 350 to 410 nm. In some cases,deprotection can be performed at 350 to 375 nm. In other cases,deprotection can be performed at 360 to 370 nm or, in some cases, ataround 365 nm.

The support can be non-transparent or transparent for light in the rangefrom UV- to the IR-light. Preferably, the support has at least 50% lighttransmission, preferably 60% light transmission, and more preferably 75%light transmission in the range from UV- to the IR-light. The supportcan have at least 50%, at least 60%, and at least 75% light transmissionat a wavelengths of 350 to 410 nm. In some cases, the support can haveat least 50%, at least 60%, and at least 75% light transmission atwavelengths of 350 to 375 nm. In some cases, the support can have atleast 50%, at least 60%, and at least 75% light transmission atwavelengths of 360 to 370 nm. In some cases, the support can have atleast 50%, at least 60%, and at least 75% light transmission atwavelengths of about 365 nm.

The washing step of the support between the capping step and thedeprotection step of the method for synthesis of an oligopeptidemicroarray can be optional. Preferably there are one or more washingsteps between the capping step and the deprotection step of the methodfor synthesis of an oligopeptide microarray. Washing of the support isperformed by a polar organic solvent or a mixture of organic solvents.

Synthesis of the oligopeptide microarrays can be performed usingphotolithography-based techniques. Therefore, a photolithographic maskis used to expose respective features to light in order to deprotect thefunctional groups, preferably the alpha-amino groups of the peptides,for coupling of the next amino acid. Preferably, masklessphotolithography is used to direct light onto respective features on anoligopeptide microarray. For this purpose, maskless photolithographyuses controllable devices, e.g., computer controlled devices, which haveindividually addressable elements to direct light onto respectivefeatures. Such controllable devices are selected from, but not limitedto, light-transmissive LCD displays and beam splitters. Preferably, adigital micro mirror device is used as a controllable device, which isan array of individually addressable aluminum mirror elements that areoperable under software control. Such elements redirect light ontorespective features on a microarray. Most preferred as a micro mirrordevice is the Digital Light Processor (DLP) from Texas Instruments, Inc.

Photoirradiation can be spectrally limited to wavelengths of 350 to 410nm, preferably to wavelengths of 350 to 375 nm, more preferably towavelengths of 360 to 370 nm, much more preferably to wavelengths of 363to 367 nm and most preferred wavelengths of 365 nm.

Spatial resolution by directing light onto respective features overindividually addressable aluminum micro mirrors may lead to manydensities of choice of oligopeptides per surface area. The microarraycan have a density of at least 10,000 and preferably at least 50,000oligopeptide features per cm². Applied to the area of a microarray, asingle microarray of 75×25 mm size may contain at least 385,000 uniqueoligopeptide features, preferably at least 720,000 unique oligopeptidefeatures, more preferably at least 2.1 million unique oligopeptidefeatures.

The oligopeptides synthesized on the microarray can have any length andcan contain any number of the same or of different amino acid residues.Preferably, the oligopeptides synthesized on the microarray have atleast 35 amino acid residues, more preferably the oligopeptidessynthesized on the microarray have at least 25 amino acid residues,preferred are oligopeptides synthesized on the microarray consisting of6 to 24 amino acids and preferably 9 to 18 amino acids.

Photoirradiation can be performed in the presence of an organic solvent,preferably a polar organic solvent. In some cases, photoirradiation isperformed in the presence of a polar organic solvent or a mixture ofsolvents, selected from a group consisting of, but not limited to,dimethylsulfoxide, n-methyl-2-pyrrolidone, dimethylformamide,acetonitrile, methanol, ethanol and propanol.

Deprotection, especially by photoirradiation, can be performed in theabsence and in the presence of a base. Suitable bases include carbonatesalts, ammonium salts, phosphates, thiolate salts, hydroxides, hydrides,heterocyclic amines, disilylamides, trialkylamines, bicyclic amines,organic acid salts and nitrogen-containing bases. The base in whichphotoirradiation is performed can be selected from either hydrazine,hydroxylamine or imidazole. Most preferred are weak basic, yetnucleophilic and weak reducing bases.

Methods used for the synthesis of oligopeptides or oligopeptidemicroarrays are designed in repeating cycles, comprising the basic stepsof coupling, optionally capping, optionally washing and deprotecting.During each cycle another amino acid is coupled to the oligopeptide.Therefore, the number of cycles is determined by the length of thesynthesized oligopeptides. Each step has a defined duration dependent onthe velocity of the associated chemical reaction. One limiting factorconcerning the synthesis of oligopeptides or oligopeptide microarrays isthe deprotection step together with the coupling step. Cleaving off theprotecting group by light exposure depends on the one hand on physicalparameters, such as pH, temperature, salt content, light intensity andwavelengths. On the other hand cleaving off the protecting group bylight exposure depends on, which amino acid is used in the respectivecycle in conjunction with which protecting group. For example, thedeprotection time of NPPOC-protected tyrosine is increased by a factorof 3 to 4 as compared to the remaining natural amino acids. Thus,NPPOC-protected tyrosine is the major time limiting factor of thesynthesis of oligopeptides or oligopeptide microarrays. In contrast, thedeprotection time of Benzoyl-NPPOC protected tyrosine is on the samelevel as the remaining natural amino acids protected with NPPOC. Thus,using Benzoyl-NPPOC protected tyrosine together with the remaining 19natural amino acids protected with NPPOC leads to the removal of themajor time limiting factor of the synthesis of oligopeptides oroligopeptide microarrays and thus to a significant increase in velocity.Coupling steps of each synthesis cycle can be less than 15 minutes, lessthan 10 minutes, or less than 5 minutes.

It is essential to have an active alignment of the microarray and theoligopeptide features, respectively, to the optical part between thesynthesis cycles in order to ensure the light exposure solely on therespective features. Therefore, it is necessary to adjust the positionof the oligopeptide microarray over a duration of over 36 hoursaccurately in one and the same position with a tolerance of about 1 μm.To achieve this goal, the oligopeptide array and the micro mirror arrayare both actively aligned by a control system. Positioning ofphotoirradiation beams onto the support can be controlled and adjustedover time. In some cases, positioning of photoirradiation beams onto thesupport can be controlled and adjusted before at least each 4^(th)irradiation (deprotection step) or, in some cases, before eachirradiation.

The support can be made of any material known by the skilled person usedfor the synthesis of an oligopeptide microarray, preferably the supportis made of plastic, glass, carbon on glass, metal on glass, or plasticon glass. Preferably, plastic is used as a support. Most preferred is aplastic solid support. Adjustment can be performed by means of adjustingthe position of the plastic solid support.

The microarray can be located on a support, preferably a plastic solidsupport, comprising at least 10,000 and preferably at least 50,000oligopeptide features per cm². Applied to the area of a microarray, asingle microarray may contain at least 385,000 unique oligopeptidefeatures, preferably at least 720,000 unique oligopeptide features, morepreferably at least 2.1 million unique oligopeptide features.

The C-terminal amino acid residues of the oligopeptide microarray arecovalently bound to the surface of the support, preferably a plasticsolid support, via peptide bonds. The C-terminal amino acids of theoligopeptides can also be coupled to the surface of the support,preferably a plastic solid support, with an ε-amino-hexanoic-acid linkermoiety.

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES Example 1 Creation of CCDS Proteome Design

The collection of protein sequences was downloaded from the ConsensusCoding Sequence (CCDS) database (available atncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi on the World Wide Web; archiveCCDSprotein.20090902.faa.gz). This set contained 23,754 proteinsequences, totaling 13,405,531 amino acids (aa). A custom perl scriptwas used to generate 12-mer oligopeptides from each protein sequence, atan interval of 6 aa. The number of synthesis cycles necessary tosynthesize each oligopeptide was evaluated using the following aminoacid sequence: A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y,V. Oligopeptides that could not synthesized using 7 repetitions of thepreceding sequence were truncated to a minimum length of 8 aa. If theystill could not be synthesized at that minimal length, the oligopeptidewas discarded. For this set of protein sequences, 1,931 oligopeptideswere discarded, mostly consisting of long runs of single amino acids. Anadditional 39 oligopeptides were discarded because they contained codesfor non-standard amino acids. After discarding these 1,970oligopeptides, a set of 2,198,610 oligopeptides remained. With theseoligopeptide deletions, only 22 proteins were completely eliminated fromthe CCDS dataset.

Roche NimbleGen's array layout software, ArrayScribe, was used torandomly distribute the oligopeptides across the array design template.The final array design consisted of three 1050 feature wide×1400 featurehigh subarrays. Each feature was 13.67 μm square. The oligopeptides werearranged randomly in a checkerboard fashion in each subarray, such thata maximum of 735000 oligopeptides could be synthesized on each subarray.After placement of the oligopeptides, a series of files was produced todirect the light-mediated deprotection of individual features for eachamino acid synthesis cycle.

Example 2 Creation of the SwissProt/RefSeq Proteome Design

A set of human protein sequences was retrieved from UniProt (uniprot.orgon the World Wide Web), consisting of 40,035 protein sequences, totaling23,843,970 amino acids. Due to the large content size, several filteringsets were implemented to reduce the content size, enriching for moreinformative oligopeptides. The first step was to mask the proteinsequences for low-complexity regions. This was done using the publiclyavailable segmasker application (v1.0.0 from blast 2.2.23 package) fromNCBI. The default parameters were used to mask low-complexity regions; atotal of 2,258,766 aa were masked. A custom perl script was used togenerate 16-mer oligopeptides from each protein sequence, at an intervalof 6 aa. The number of synthesis cycles necessary to synthesize eacholigopeptide was evaluated using the following amino acid sequence: A,R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V. Oligopeptidesthat could not synthesized using 10 repetitions of the precedingsequence were truncated to a minimum length of 10 aa. If they stillcould not be synthesized at that minimal length, the oligopeptide wasdiscarded. For this set of protein sequences, only 5 oligopeptides werediscarded for length consideration, because the full protein sequencewas shorter than 10 amino acids. A total of 641,066 oligopeptides werediscarded due to low-complexity masking. An additional 761 oligopeptideswere discarded because they contained codes for non-standard aminoacids. After discarding these 641,832 oligopeptides, a set of 3,288,695oligopeptides remained. With these oligopeptide deletions, only 85proteins were completely eliminated from the CCDS dataset. This set ofoligopeptides contained 1,462,415 unique oligopeptides and 684,075oligopeptides shared by two or more proteins. These shared oligopeptidessequences were placed on the array only once, and a correspondence keygenerated to indicate the original sequence identifiers and positionsfor later data analysis.

Roche NimbleGen's array layout software, ArrayScribe, was used torandomly distribute the final 2,146,490 oligopeptides across the arraydesign template. The final array design consisted of three 1,050 featurewide×1,400 feature high subarrays. Each feature was 13.67 μm square. Theoligopeptides were arranged randomly in a checkerboard fashion in eachsubarray, such that a maximum of 735,000 oligopeptides could besynthesized on each subarray. After placement of the oligopeptides, aseries of files was produced to direct the light-mediated deprotectionof individual features for each amino acid synthesis cycle.

Example 3 Synthesis, Binding and Analysis of CCDS Proteome Arrays

Arrays were synthesized on Greiner Bio One HTA 3D amino slides. Briefly,400 μl of an amino acid (diluted to 60 mM in Dimethylformamide (DMF))was delivered to a reaction vessel and activated with 200 μl ofactivator (60 mmol HBTU, 60 mmol HOBt in DMF) and 200 μl base (0.27MDIPEA in DMF). The mixture was mixed via bubbling through air.Activation proceeded for 5 minutes. The entire activated amino acidmixture was delivered to the slide in the flow cell and allowed tocouple for 1 minute. After coupling, 3000 μl of 1-Methyl-2-pyrrolidinone(NMP) was washed over the array, before flowing 1000 μl of exposuresolvent (0.01% hydroxylamine in NMP) to wash and cover the active arraysurface. Features were then deprotected by removing the NPPOC protectinggroup from the amino terminus of the bound amino acid. Deprotectionoccurred in exposure solvent (see above) through the delivery of 365 nmUV light from a mercury-xenon lamp at an approximate power of 161 mw/cm²for 60 seconds. Initial coupling of a 6-hexanoic acid linker as abovewas followed by 143 sequential peptide coupling and directedphotodeprotection steps to build the desired peptide sequences. Eachpeptide had an additional Serine residue at each end in order to improvehydrophilicity of the support bound peptide and enable easy access ofantibodies.

After completion of synthesis arrays were washed in 3000 μl of MeOH, anddried with argon. Peptide side-chain removal was accomplished byimmersion in a 95% TFA/4.5% H₂O/0.5% Triisopropylsilane solution for 30minutes at room temperature. Arrays were then washed twice in copiousamounts of MeOH, rinsed with H₂O, and dried with an argon jet.

Binding of arrays was proceeded by washing of arrays in 1×TBSTT (0.055%Tween-20 and 0.22% Triton X-100) for 2 minutes, followed by washing in1×TBS for 2 minutes. After washes, arrays were incubated overnight (˜16hours) in binding solution (22.5 μl 1×TBS, 18 μl DiH₂O, 4.5 μl 5%alkali-soluble Casein) with 1:15000 final dilution of polyclonalanti-ADA produced in rabbit (Sigma-Aldrich HPA001399 0.19 mg/ml),polyclonal anti-PAPOLA produced in rabbit (Sigma-Aldrich HPA001788, 0.05mg/ml) and monoclonal anti-poly-Histidine (Sigma-Aldrich H1029, 0.25mg/ml) produced in mouse. Incubation occurred at ambient temperaturewith mixing via a Roche NimbleGen Hybridization System.

After removal from primary incubation, arrays were washed twice for 2minutes each in 1×TBSTT and once in 1×TBS for 2 minutes. Arrays werethen placed in an opaque plastic staining jar containing 75 ml bindingsolution (see above) containing a 1:10000 final dilution of donkeyanti-rabbit IgG (Jackson ImmunoResearch 711-165-152, 1.5 mg/ml)Cy3-labeled secondary antibody for detection of bound ADA and PAPOLA anda 1:10000 dilution goat anti-mouse (Invitrogen A10521, 2 mg/ml)Cy3-labeled secondary antibody. Incubation occurred with gentle shakingat ambient temperature for 3 hours.

After secondary binding, arrays were washed twice for 2 minutes in1×TBSTT, rinsed with DiH₂O, and dried with an argon stream.

Arrays were scanned using a Roche NimbleGen MS200 scanner, in the 532 nmchannel at a resolution of 2 μm. Images were analyzed using NimbleScan,raw intensities of the features were plotted vs. protein position usingSignalMap software from Roche Nimblegen.

Example 4 Binding of Anti-PAPOLA Polyclonal Antisera to OligopeptideArrays

Targeted peptide arrays were designed utilizing similar tiling strategyand software as described in Example 1, and synthesized in similarfashion as detailed Example 3. The targeted arrays were designed from atarget population of 52 proteins including the full PAPOLA proteinsequence and an antigen subset (the “PA52 design”), tiled at singleamino acid resolution (i.e., a 1 amino acid “step”). Binding experimentsusing ATLAS (Sigma-Aldrich) derived anti-PAPOLA polyclonal antibodiesfor primary antibody binding was performed as described in Example 3above. Secondary antibody binding was performed using donkey anti-rabbitIgG Cy3-labeled secondary antibody for detection of bound PAPOLAantibodies.

FIG. 3A shows the fluorescence detected using a PA52 design microarrayfor a target subset of PAPOLA protein comprised of approximately 140amino acids, tiled at single amino acid resolution (1×10 tiling). Thisbinding experiment resulted in identification of three potentialepitopes:

(SEQ ID NO: 2) NSSGSSQGRNSPAPAVTA (9 features, positions 38-46)(SEQ ID NO: 3) NAATKIPTPIVGV (4 features, positions 126-129)Possibly a third epitope at (SEQ ID NO: 4) ATQPAISPPPKP(3 features, positions 88-90).

FIG. 3B shows a trace of the same array (PA52 design), viewing the fulllength PAPOLA protein (approximately 730 amino acids), tiled at singleamino acid resolution. When the data is examined at this level, threepotential epitopes are again identified:

(SEQ ID NO: 5) NSSGSSQGRNSPAPAVTA (9 features, positions 548-556)(SEQ ID NO: 6) NAATKIPTPIVGVK (4 features, positions 637-640)Possibly a third epitope at (SEQ ID NO: 7) ATQPAISPPPKP(3 features, positions 599-601)

FIG. 3C shows an example of polyclonal anti-PAPOLA binding to PAPOLAtarget on a CCDS “full proteome” microarray designed and synthesized insimilar fashion to Example 1 and Example 3 above. This oligopeptidemicroarray is a 6×12 design (i.e., 12-mer oligopeptides with a 6meroverlap or “step”). Identified epitopes from this experiment are:

(SEQ ID NO: 8) LNSSGSSQGRNSPAPAVT (two features, position 547, 553)(SEQ ID NO: 9) NAATKIPTPIVG (position 637) Possibly a third epitope at(SEQ ID NO: 10) QPAISPPPKP (position 601).Results of all three scans exemplified in FIGS. 3A-C are found in Table1:

TABLE 1 Epitope Epitope Epitope PA52_sub 1aa_tile NSSGSSQGRNSPAPAVTANAATKIPTPIVGV ATQPAISPPPKP (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4)PA52_full 1aa_tile NSSGSSQGRNSPAPAVTA NAATKIPTPIVGVK ATQPAISPPPKP(SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) CCDS_proteome_6aa_tileLNSSGSSQGRNSPAPAVT NAATKIPTPIVG QPAISPPPKP (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 10)

From this, one can discern that the full proteome design of the presentinvention, using a 6×12 tiling design, can detect essentially the samepotential epitopes for anti-PAPOLA binding as are demonstrated by arrayswith significantly less content and much higher amino acid resolution.This utility of a full proteome array provides unprecedented opportunityto screen for antibody binding across an essentially complete proteome,and allows for determination of the epitopes against which an antibodybinds. Further, this provides opportunity to determine if a particularantibody exhibits high cross-reactivity to a large number of epitopesthroughout the proteome, or if the antibody's reactivity is morespecifically confined to expected epitopes or targets.

Example 5 Synthesis of the Antigen Sequences of 52 Selected Antibodies

Fifty-two antibodies were selected from the Human Protein Atlas database(proteinatlas.org on the World Wide Web). The respective antigensequences were synthesized according to Example 3 in 164 successivesynthesis cycles as 10-mer peptides with an overlap of 9 amino acids. Insome cases, the overlap was shorter, but none was less than 6 aminoacids. Deprotected oligopeptide arrays were incubated with mixtures ofantibodies at dilution factors according to the data sheetrecommendation for WESTERN analysis in the manufacturer's recommendedbuffer and their binding epitopes were determined as the highestintensity signals upon fluorescent labeling with an anti-rabbitsecondary antibody (see FIG. 4). In some cases, the arrays were washedwith buffer containing 0.1% SDS in order to remove non-specific boundantibodies.

Example 6 Sensitivity Titration of Anti-PAPOLA in Buffer

A peptide array manufactured in similar fashion as described in Example1 and Example 3 above was incubated with escalating dilutions of theanti-PAPOLA antibody (PolyA Polymerase alpha, HPA001788), from 1:10,000up to 1:240,000 in discrete sub-arrays formed by a Roche Nimblegen12-plex sample chamber assembly. Staining and washing was performed asdescribed above. FIG. 6 depicts the binding results for variousdilutions of antibody, focusing on amino acid positions 126-129 of thePAPOLA antigen sequence. As depicted in FIG. 5, raw data afterfluorescent scanning with a Roche Nimblegen MS 200 micro array scannerrevealed the epitopes clearly visible even at the highest dilution,comparing to an antibody concentration of about 200 pg/mL, with arelative signal to noise ratio of 4:1.

Example 7 Oligopeptide Array Binding With A Serum Sample

An isolated IgG pool from a colorectal cancer serum sample was bound toan oligopeptide array containing approximately 40,000 12-meroligopeptide features with an 1′-mer overlap. The serum sample was froma serum bank containing clinically characterized sera for differentdiseases. Samples from cancer patients were screened with an anti-p53assay for high-titer sera. From a high-titer serum, IgG was isolated bystandard procedures. A dilution (10 μg/ml) of this preparation was usedfor binding studies. Incubation of array with sample was performedovernight in a Maui/X1 mixer at room temperature. After incubation, thearray was washed for two minutes each with TBSTT, TBST/TBS, and H₂O.Secondary antibody staining was performed using Goat(anti)Human (JacksonImmunoResearch) labeled with Cy3 dye, (1:12,500) for 3 hours. The arraywas washed for two minutes each with TBSTT, TBST/TBS, and H₂O, dried andscanned in an MS200 scanner.

FIG. 6 shows the data generated by this scan. The data shows intensebinding to the peptides representing P-53 protein, a known antigen inautoimmunity in cancer. This data trace also suggests that there aremultiple binding events from many auto-antibodies in a single patient.In FIG. 6, the oligopeptides that are tiling positions 10-40(approximately) are peptides that represent a known antigen. In ELISAtesting, the sample used in FIG. 6 tested positive against that specificantigen; however, FIG. 6 demonstrates significant binding events atother loci within the P-53 protein. In fact, the other loci in thispatient sample show significantly higher intensity binding than thebinding of the known antigenic site.

In current practice, a patient who is already showing signs of illness(e.g., a patient who is already suspected of having cancer) maydemonstrate auto-immunity to certain known antigens, which isconfirmatory of the cancer diagnosis. However, if the currently knownauto-immunity test comes back negative, the clinician is still not wellinformed as the autoimmunity antibodies may not be highly reactive tothe known (currently used) antigens. Autoimmune antibodies can be foundin cancer patients almost all the time at a stage when the patient isalready presenting with clinical signs of illness; however, currenttests may not be as complete for diagnosis or early detection as a toolthat provides a broader look at the binding events, such as the presentinvention. These data demonstrate that looking at autoimmune antibody“fingerprints” across a broader spectrum of potential antigenic sitesmay improve the prognostic value of autoimmunity tests.

Example 8 Serum Binding Assay Using Peptide Proteome Array

This process is identical to procedure in Example 3, with the followingexceptions:

1. Anti-ADA, PAPOLA, and poly-Histidine antibodies are replaced with a1:20 dilution of serum sample.

2. Secondary binding is performed with appropriate secondary antibody(i.e. species-specific)

3. Post-secondary binding washes will include 0.2% SDS wash for 2 hoursor longer

Arrays of specified design (CCDS proteome, RefSeq proteome, SwissProtproteome) are synthesized and side-group deprotected as above.

Binding of arrays is proceeded by washing of arrays in 1×TBSTT (0.055%Tween-20 and 0.22% Triton X-100) for 2 minutes, followed by washing in1×TBS for 2 minutes. After washes, arrays are incubated overnight (˜16hours) in binding solution (22.5 μl 1×TBS, 18 μl DiH₂O, 4.5 μl 5%alkali-soluble Casein) with 1:20 final dilution of serum sample.Incubation occurs at ambient temperature with mixing via a RocheNimblegen Hybridization System.

After removal from primary incubation, arrays are washed twice for 2minutes each in 1×TBSTT and once in 1×TBS for 2 minutes. Arrays are thenplaced in an opaque plastic staining jar containing 75 ml bindingsolution (see above) containing a 1:10000 final dilution of appropriate,species-specific, fluorescently labeled secondary antibody. Incubationoccurs with gentle shaking at ambient temperature for 3 hours.

After secondary binding, arrays are washed twice for 2 minutes in1×TBSTT. Arrays are then subjected to stringent wash in 0.2% SDS for 2hours with gentle shaking, prior to final rinse with DiH₂O and dryingwith argon.

Arrays are scanned using a Roche Nimblegen MS200 scanner, in theappropriate channel (e.g. 532 nm, 635 nm) at a resolution of 2 μm.Images are analyzed using NimbleScan, raw intensities of the featuresare plotted vs. protein position using SignalMap.

Example 9 Binding of Anti-ADA Polyclonal Antisera to Oligopeptide Arrays

Targeted peptide arrays were designed utilizing similar tiling strategyand software as described in Example 1, synthesized and bindingexperiments performed as described in Example 3. FIGS. 7A, 7B and 7Cshow the results of binding experiments using ATLAS (Sigma-Aldrich)derived anti-ADA polyclonal antibodies for primary antibody binding asdescribed in Example 3 above. Secondary antibody binding was performedusing donkey anti-rabbit IgG Cy3-labeled secondary antibody fordetection of bound ADA antibodies.

FIG. 7A shows the fluorescence detected using a PA52 design microarrayfor a target subset of ADA protein comprised of approximately 140 aminoacids, tiled at single amino acid resolution (1×10 tiling). This bindingexperiment resulted in identification of two potential epitopes:

(SEQ ID NO: 11) SLLPGHVQAYQEAV (5 features, positions 9-13)(SEQ ID NO: 12) HTLEDQALYNRLQEN (6 features, positions 58-63)

FIG. 7B shows a trace of the same array (PA52 design), viewing the fulllength ADA protein, tiled at single amino acid resolution. When the datais examined at this level, two potential epitopes are again identified:

(SEQ ID NO: 13) SLLPGHVQAYQEAV (5 features, positions 192-196)(SEQ ID NO: 14) HTLEDQALYNRL (4 features, positions 241-243)

FIG. 7C shows an example of polyclonal anti-ADA binding to ADA target ona CCDS “full proteome” microarray designed and synthesized in similarfashion to Example 1 and Example 3 above. This oligopeptide microarrayis a 6×12 design (i.e., 12-mer oligopeptides with a 6mer overlap or“step”). The identified epitope from this experiment is:

(SEQ ID NO: 15) LLPGHVQAYQEA (1 feature, position 193)

The expected epitope binding at approximately position 241 was missingin the full proteome binding experiment; however, this was readilyexplained by the fact that the features at and around position 241 wereoccluded by a slide logo mark indicating the slide manufacturer. Thefeature that was expected to show a peak at position 241 instead showedan actual drop below background levels, thus demonstrating that thefeature was compromised because of the logo position.

From these data, one can discern that the full proteome design of thepresent invention, using a 6×12 tiling design, can detect essentiallythe same potential epitopes for anti-ADA binding as are demonstrated byarrays with significantly less content and much higher amino acidresolution.

Example 10 Binding of a Monoclonal Anti-Poly-Histidine Antibody to FullProteome Array

FIG. 8 shows an example of monoclonal anti-poly-Histidine antibodybinding to a potential epitope on a CCDS “full proteome” microarray.This microarray was designed and synthesized and binding experimentsconducted as described in Example 1 and Example 3 above.

This oligopeptide microarray is a 6×12 design (i.e., 12-meroligopeptides with a 6-mer overlap or “step”). The identified epitopefrom FIG. 8 is:

(SEQ ID NO: 16) HFQHHHHH (1 feature, position 511)

This is only one example from this binding experiment: there werenumerous other examples in the design where the monoclonalanti-poly-Histidine antibody bound other peptides with poly-Histidinestretches (data not shown). This binding experiment demonstrated that afull proteome array can readily be used for binding of monoclonalantibodies as well as polyclonal antibodies.

What is claimed is:
 1. A microarray comprising at least 50,000oligopeptide features per cm² wherein the features represent betweenabout 90% and 100% of a target proteome, the target selected from avirus and an organism, and wherein at least a portion of the featurescomprise oligopeptides having a terminal2-(2-nitro-4-benzoyl-phenyl)-propoxycarbonyl(benzoyl-NPPOC)-protectedtyrosine residue.
 2. The microarray of claim 1, comprising at least100,000 oligopeptide features per cm².
 3. The microarray of claim 1,comprising at least 200,000 oligopeptide features per cm².
 4. Themicroarray of claim 1, wherein the organism is human.
 5. The microarrayof claim 1, wherein substantially all of the oligopeptides are the samelength.
 6. The microarray of claim 1, wherein substantially all of theoligopeptides are 9 to 18 amino acid residues in length.
 7. Themicroarray of claim 6, wherein substantially all of the oligopeptidesare 10 to 15 amino acid residues in length.
 8. The microarray of claim7, wherein substantially all of the oligopeptides are 12 amino acidresidues in length.
 9. The microarray of claim 1, wherein eacholigopeptide feature overlaps in amino acid sequence with the amino acidsequence of at least one other feature by at least 3 contiguous aminoacid residues.
 10. The microarray of claim 9, wherein each oligopeptidefeature overlaps by at least 9 amino acid residues.
 11. A method forsynthesizing a microarray comprising at least 50,000 oligopeptidefeatures per cm² wherein the features represent between about 90% and100% of a target proteome, the target selected from a virus and anorganism, the method comprising synthesizing the oligopeptide featureson the microarray such that an oligopeptide feature overlaps in aminoacid sequence with the amino acid sequence of at least one otheroligopeptide feature by at least one amino acid residue, wherein atleast a portion of the features comprise oligopeptides having a terminal2-(2-nitro-4-benzoyl-phenyl)-propoxycarbonyl(benzoyl-NPPOC)-protectedtyrosine residue.
 12. The method of claim 11, wherein each oligopeptidefeature overlaps in amino acid sequence with the amino acid sequence ofat least one other oligopeptide feature by exactly 9 amino acidresidues.
 13. The microarray of claim 1, wherein the benzoyl-NPPOC iscleavably bound to the terminal tyrosine residue.
 14. The microarray ofclaim 1, wherein the benzoyl-NPPOC is photolabile.
 15. The microarray ofclaim 1, wherein an oligopeptide feature overlaps in amino acid sequencewith the amino acid sequence of at least one other oligopeptide featureby exactly 9 amino acid residues.
 16. The microarray of claim 1 whereinthe microarray comprises tiled oligopeptide features, and wherein theoligopeptides represent portions of at least about 5,000 of the proteinsexpressed in a species.
 17. The microarray of claim 16, wherein theoligopeptides represent portions of at least about 10,000 expressedproteins.
 18. The microarray of claim 16, wherein the oligopeptidesrepresent portions of at least about 20,000 expressed proteins.
 19. Themicroarray of claim 16, wherein the oligopeptides represent portions ofbetween about 5,000 and 50,000 expressed proteins.
 20. The microarray ofclaim 15, wherein the species is human.